The initiation and persistence of inflammation is tightly regulated by cell surface receptors on immune cells that function to both amplify the immune response and clear the offending agent. The persistence of inflammation may result in disease inclusive of autoimmune, chronic allergies and asthmatic diseases. One common feature of these diseases is the central role of Fc receptors and immune complexes in the persistence of disease. A second feature is the persistence of high levels of cytokines which serve to recruit other immune cells amplifying the response. Our interest is in understanding how Fc receptors communicate with the nucleus to initiate cytokine responses. The immediate objective is to identify the molecular events that activate cytokine gene transcription and to evaluate their suitability as targets in therapeutic intervention of disease. We chose to work with mast cells because they are key participants in many inflammatory responses, they express the well-characterized high affinity receptor for IgE (Fc epsilon RI), and they respond to immune complex occupation of the Fc epsilon RI by producing and secreting a diverse array of cytokines and other allergic mediators. Our prior work identified two major families of proteins that link the Fc epsilon RI to nuclear events. The first is the protein kinase C family which is comprised of twelve serine/threonine kinases of which five are known to be expressed in mast cells. Our studies demonstrated that of the five distinct isoforms found in mast cells, two isoforms, beta and epsilon, specifically regulate the synthesis of the AP-1 constituents c-fos and c-jun. One isoform, PKC delta, phosphorylates the Fc epsilon RI and may serve to regulate the receptors ability to communicate intracellularly. These results demonstrate that the different PKC isoforms have specific functions in mast cell responses and thus, makes it more likely that therapeutic targeting of a specific isoform(s) that regulates cytokine gene expression may be possible. The second family of proteins we are studying is that of guanine nucleotide exchange factors (GEF). These proteins facilitate the exchange of guanine diphosphate (GDP) to guanine trisphosphate (GTP) from small GTP binding proteins which serves to activate the latters function in regulating numerous signaling pathways. We found that a member of the GEF family called Vav, which is expressed only in hematopoietic cells, may link the Fc epsilon RI to nuclear events, since it is activated in these cells upon Fc epsilon RI stimulation and serves to activate mitogen-activated protein kinase (MAPK) family members that are known to stimulate gene transcription. Prior evidence from our lab and and that of others demonstrated that Vav contributes to the activation of cytokine gene transcription, in particular to the induction of interleukin-2 (IL-2). As a hematopoietic cell-specific protein, Vav may serve a unique role in immune cells, and investigating its functional role in these cells may identify new areas with therapeutic potential. In the past year our objectives were: 1. To develop a method amenable to rapid analysis of function for multiple wild type and mutant proteins expressed in mast cells or in any other poorly transfected cell, in order to understand the role of these proteins in signal transduction. 2. To determine the significance of the Fc epsilon RI phosphorylation mediated by PKC delta to mast cell responses. 3. To determine if the Fc epsilon RI-mediated production of TNF-alpha by mast cells is regulated by NF-kappa B. 4. To study the role of Vav in the induction of cytokine gene expression. 5. To determine where Vav is localized in the cell and the importance of this localization to Vav function. Our findings showed that Vav stimulates an increase in IL-2 mRNA in mast cells, an observation previously reported for the role of Vav in T-cells. However, novel to our studies is the role of Vav in stimulating large increases in mRNA for IL-6. This increase in IL-6 mRNA is dependent on the activation of c-jun N terminal kinase (JNK) by Vav via the activation of Rac 1 by Vav. We also found that the small GTP-binding protein Ras does not contribute to the Vav-mediated induction of IL-6. Thus, these findings show that Vav/JNK activity is important to the production of IL-6. In addition, we used confocal microscopy to localize the cellular site of Vav activity to the plasma membrane. In response to Fc epsilon RI-stimulation we found that Vav redistributes from the cytoplasm to a submembraneous location forming small micro-aggregates which co-localize with the Fc epsilon RI. The localization of Vav to these sites is dependent on the SH2 domain of Vav (a protein domain that recognizes proteins phosphorylated on tyrosine residues) and this localization is important to Vav's ability to activate JNK. In other studies we identified the site of phosphorylation of the Fc epsilon RI gamma chain by PKC delta and found that mutation of this threonine 60 (T60) to alanine resulted in a defective Fc epsilon RI mediated TNF-alpha production and in the inhibition of antigen clearance from the mast cell surface. In the analysis of affected signaling pathways we found that mast cells with the T60A mutated gamma chain were defective in activation of Syk kinase, a kinase essential to mast cell degranulation and cytokine production. These findings demonstrate that the activation of Syk kinase is dependent on the threonine phosphorylation of the Fc epsilon RI gamma chain in these cells and identify a new area with therapeutic potential in allergic diseases. In conclusion, our studies resulted in the development of a viral-based expression system to study protein function in cells that are normally difficult to transfect or infect. We identified the site of Vav function in mast cells and found that it specifically regulates IL-6, a cytokine important in inflammatory disease. We also identified a novel NF-kappa B like activity in mast cells that may be mast cell specific and is essential to the induction of TNF-alpha gene expression.